Apoptosis-associated speck-like protein containing a CARD regulates the growth of pancreatic ductal adenocarcinoma

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is a key adaptor protein of inflammasomes and a proapoptotic molecule; however, its roles in signal transduction in pancreatic ductal adenocarcinoma (PDAC) cells remain unknown. Here, we clarified the role and mechanisms of action of ASC in PDAC using clinical evidence and in vitro data. ASC expression in PDAC tissues was analyzed using public tumor datasets and immunohistochemistry results of patients who underwent surgery, and PDAC prognosis was investigated using the Kaplan–Meier Plotter. ASC expression in PDAC cells was downregulated using small-interfering RNA, and gene expression was assessed by RNA sequencing. Review of the Oncomine database and immunostaining of surgically removed tissues revealed elevated ASC expression in PDAC tumors relative to non-tumor tissue, indicating poor prognosis. We observed high ASC expression in multiple PDAC cells, with ASC silencing subsequently inhibiting PDAC cell growth and altering the expression of cell cycle-related genes. Specifically, ASC silencing reduced cyclin D1 levels and stopped the cell cycle at the G1 phase but did not modulate the expression of any apoptosis-related molecules. These results show that ASC inhibited tumor progression via cell cycle modulation in PDAC cells and could be a potential therapeutic target.

Patients and pancreas specimens. PDAC specimens and adjacent normal pancreatic tissues were obtained from patients who underwent surgery at the Ehime University Hospital. All the specimens used in this study were obtained from eight samples collected between 2018 and 2019 from PDAC patients. Written informed consent was obtained from all enrolled participants, and the study protocol conformed to the ethical guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Ehime University Hospital (approval no. 1712007).
Immunohistochemistry (IHC) of PDAC specimens. Pancreatic tissues were fixed in formalin, sections (3-µm thick) were cut from each block, and adjacent sections were stained using IHC. Paraffin-embedded samples were dewaxed and rehydrated, and antigen retrieval was performed by autoclaving for 1 min at 110 °C in citrate buffer (pH 6.0). Endogenous peroxidase activity was inactivated by incubation with methanol containing 1% hydrogen peroxidase for 20 min. The sections were then incubated in 1% blocking goat serum for 30 min to reduce nonspecific reactions. For IHC, the sections were probed overnight with the anti-ASC antibody (1:200; 10500-1-AP; Proteintech, Tokyo, Japan) at 4 °C. The tissue sections were then incubated with a peroxidaselabeled secondary antibody (Histofine Simplestain Max POR; Nichirei, Tokyo, Japan) for 1 h at room temperature and then with Simple Stain DAB solution (Nichirei). Photomicrographs were obtained using a Nikon ECLPSE 50i with a Nikon digital camera (DS-F11; Nikon, Tokyo, Japan). Staining intensity was scored with no prior knowledge of clinicopathological results, in the following manner: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. Immune-reaction intensity (optical density, OD) was examined using ImageJ (NIH, Bethesda, MD, USA) and calculated as OD = log [max intensity/mean intensity (max intensity = 255]. The Kaplan-Meier Plotter. The Kaplan-Meier Plotter (https:// kmplot. com/ analy sis/) is a web server for the discovery and validation of survival biomarkers 16 . We used the survival-analysis tool in the Kaplan-Meier Plotter to analyze the prognostic significance of ASC mRNA. Survival analysis was performed using the Kaplan-Meier method, and a hazard ratio (HR) with 95% confidence intervals (CIs) and a log-rank P-value were calculated. Kaplan-Meier Plotter was also used to compare the predictive value of several gene combinations in patients with low and high mutational burden.
Cancer Cell Line Encyclopedia (CCLE) analysis. ASC mRNA levels in a series of cancers were analyzed using the CCLE (https:// porta ls. broad insti tute. org/ ccle/ home). According to the website, raw microarray data of PDAC cell lines were converted to a single value for each probeset using the Robust Multi-array Average algorithm and quantile normalization.
Cells and culture conditions. Four PDAC cell lines (PANC-1, BxPC-3, AsPC-1, and MIA PaCa-2), which were initially generated using samples from PDAC patients, and hTERT-HPNE cells were obtained from the American Type Culture Collection (Manassas, VA, USA). PANC-1 and MIA PaCa-2 cells were cultured in Dulbecco's modified Eagle medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies) and 1% penicillin. BxPC-3 and AsPC-1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 1% penicillin. hTERT-HPNE cells were cultured in 75% DMEM and 25% Medium M3 Base with 5% FBS, 10 ng/mL human recombinant epidermal growth factor, 5.5 mM d-glucose, and 750 ng/mL puromycin. Microphotographs were obtained after various treatments using an inverted microscope (AxioVertA1 FL; ZEISS, Oberkochen, Germany) equipped with an Axiocam MRm camera (ZEISS). RNA extraction, cDNA synthesis, and real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and the RNA was reverse transcribed using RT-PCR kits (Applied Biosystems, Tokyo, Japan). Real-time PCR was performed using a LightCycler 480 (Roche, Tokyo, Japan) with SYBR Green I (Roche). Sequences of the primers used to amplify human genes are indicated in Table S1. Glyceraldehyde 3-phosphate dehydrogenase served as an internal reference gene, and the relative change was calculated by relative quantification using the formula 2 −ΔΔCt .
RNA sequencing (RNA-seq) and data analyses. Total RNA was collected from PANC-1 and AsPC-1 cells at 72 h after transfection with either control siRNA, ASC siRNA (1), or ASC siRNA (2), and the integrity of isolated RNA was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA samples with an RNA integrity number greater than eight were normalized to 100 ng/μL before further analyses. RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA sample prep kit setA according to manufacturer instructions (Illumina, San Diego, CA, USA) and subsequently validated to ensure an average size of ~ 330 to ~ 340 bp using a 2100 Bioanalyzer and the Agilent DNA1000 kit. Paired-end reads (75 bp) were sequenced using the MiSeq reagent kit V3 150 cycle on a MiSeq system (Illumina). Mapping to human genome data hg19 was performed using Tophat (https:// ccb. jhu. edu/ softw are/ tophat/ index. shtml), and the expression analysis tool Cufflinks (http:// cole-trapn ell-lab. github. io/ cuffl inks/) was used to normalize each sample before determining the differences in expression between control siRNA (n = 3), ASC siRNA (1) (n = 3), and ASC siRNA (2) (n = 3) using the tmm package in R (https:// www.r-proje ct. org/). Raw and processed data were submitted to the Gene Expression Omnibus (GSE168842) database. Pathway and process enrichment analyses were performed using all genes that after alignment and normalization were found to be downregulated with an adjusted p < 0.05 using Metascape 18 and gene set enrichment analysis (GSEA) 19 .
Cell cycle analysis. Cells were seeded at a density of 1 × 10 6 cells/well in 10-cm dishes. At 72 h after siRNA transfection, a BD Cycletest TM Plus DNA kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to purify the sample according to manufacturer instructions, and cell cycle distribution was evaluated by flow cytometry (Gallios flow cytometer; Beckman Coulter, Brea, CA, USA). Apoptosis assay. PANC-1 cells were transfected with control siRNA or ASC siRNA, and a Mebstain apoptosis terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) kit II (MBL, Nagoya, Japan) was used to detect oligonucleosomal DNA breaks in PANC-1 cells 3 and 5 days after transfection.
Proteome profiling was performed using commercially available human apoptosis proteome profiler arrays (R&D Systems, Minneapolis, MN, USA) according to manufacturer instructions. Expression levels of 35 apoptosis-related proteins (effector and signaling molecules) were evaluated by densitometric analyses of the arrays using ImageJ (NIH).

Cytokine quantification in tumor cells.
Tumor cells were plated onto 6-well plates in 2 mL DMEM or RPMI 1640 supplemented with 10% FBS and incubated for 24 h at 37 °C. Medium was replaced with 2 mL of fresh medium in each well, and the cells incubated for another 48 h. Supernatants were collected, and the levels of IL-1β were assessed using enzyme-linked immunosorbent assay (ELISA; human IL-1β/IL-1F2 Quantikine ELISA Kit; R&D Systems).
Statistical analysis. All statistical analyses were performed using JMP software (v.14.0; SAS Institute, Tokyo, Japan). Data are expressed as the mean and standard deviation. Differences were analyzed using Student's t-test, the Wilcoxon test, and χ 2 test. Statistical significance was defined as p < 0.05 based on a two-tailed test. Correlations between two variables were evaluated by using Pearson's correlation coefficient.

Results
ASC is highly expressed in PDAC. Examination of the Oncomine database showed that ASC mRNA was significantly upregulated in PDAC in multiple microarray datasets [20][21][22][23][24][25] (Fig. 1a). A microarray meta-analysis of these six datasets revealed that ASC mRNA was significantly upregulated in PDAC (p = 0.039). Accordingly, examination of immunostaining scores for the surgically removed tissues revealed that the immunostaining intensity was predominantly stronger in PDAC tissues relative to that in tissues surrounding the tumor (Fig. 1b,c). Quantitative assessment of the immunostaining results indicated a significantly higher OD in PDAC tissue relative to that in tissues surrounding the tumor (OD: 0.409 vs. 0.216, respectively; p = 0.0181) (Fig. 1d). Furthermore, ASC expression in ducts and acinar cells was weak in tissues distant from the tumor (Fig. 1e); however, we observed strong expression of ASC in either metaplastic or normal ducts within their nearby normal tissue relative to that in acinar tissue (Fig. 1f), and ASC expression was even stronger in cancerous lesions than surrounding tissues (Fig. 1g).  (Fig. 2a). We then determined whether ASC mRNA expression is predictive of outcomes in a cohort of tumors with low and high mutation burden. We found that elevated ASC mRNA expression was predictive of poor prognosis in a cohort of patients harboring tumors with low mutation burden (n = 83; HR = 2.78, 95% CI 1.5-5.16; log-rank p = 0.00073) (Fig. 2b), whereas high ASC mRNA expression did not contribute to prognosis in tumors with high mutation burden (n = 84; HR = 0.7, 95% CI 0.38-1.29; log-rank p = 0.25) (Fig. 2c). Based on evaluations of tumor grade, we found that elevated ASC mRNA expression was predictive of poor prognosis in a cohort of patients with grade 1 and grade 2 tumors but not grade 3 (grade 1: n = 31; HR = 1,300,997,289; 95% CI 0-inf; log-rank p = 0.0044; grade 2: n = 94; HR = 1.88, 95% CI 1.04-3.4; log-rank p = 0.033; and grade 3: n = 48; HR = 1.89, 95% CI 0.9-3.95; log-rank p = 0.087) (Fig. 2d-f). www.nature.com/scientificreports/

Downregulation of ASC expression inhibits the growth of PDAC cells.
We then examined ASC expression in PDAC cell lines, finding increased expression in PANC-1, ASPC-1, and BXPC-3 cells and decreased expression in Mia-PaCa-2 cells relative to that in the pancreatic ductal epithelial cell line hTERT-HPNE, which exhibits an acinar associated with the ductal metaplasia phenotype, according to real-time RT-PCR (Fig. 3a). Additionally, we confirmed ASC expression in PDAC cell lines using the CCLE database (Fig. S1). Additionally, western blotting identified high ASC expression in PANC-1, ASPC-1, and BxPC-3 cells, low expression in hTERT-HPNE cells, and no expression in MIA PaCa-2 cells (Fig. 3b). These findings are in agreement with those of our evaluation of human tissues, with high ASC expression observed in multiple cancer cell lines relative to normal pancreatic duct epithelial cells. To investigate the role of ASC, we knocked down ASC expression using two ASC siRNAs in PANC-1 and ASPC-1 cells, both of which exhibit relatively high ASC expression. Following confirmation of successful knockdown of ASC levels by real-time RT-PCR and western blotting (Fig. 4a,b), microscopy-based observation of cells revealed inhibited proliferation (Fig. 4c). Evaluation of cell proliferation by MTS assay confirmed this observation, and that this effect increased over time (p < 0.05) (Fig. 4d). Notably, suppressed proliferation following ASC knockdown was not observed in Mia PACA-2 or hTERT-HPNE cells (Fig. S2). These results indicated that ASC is mainly involved in PDAC cell proliferation.

ASC regulates the cell cycle in PDAC cells.
To elucidate the detailed molecular mechanism underlying ASC-mediated PDAC cell proliferation, we performed RNA-seq analysis on control PANC-1 cells and ASCsilenced PANC-1 cells. We found that the total number of RNAs in the silenced cells significantly decreased relative to the control [ASC siRNA (1) and (2): 2194 and 2364, respectively]. Additionally, we analyzed 793 RNAs that were commonly reduced in the two ASC siRNA groups using Metascape (Fig. 5a). The results showed that significantly enriched signaling pathways included the cell cycle, positive regulation of organelle organization, and cell cycle phase transition. The top 20 clusters of significantly enriched terms are shown in Fig. 5b,c. Furthermore, GSEA revealed significant relationships between the expression of cell cycle-related genes (normalized enrichment score = 1.60; and false discovery rate = 0.12; p < 0.01). (Fig. 5d). Similar data were obtained in RNAseq experiments using AsPC-1 cells (Fig. S3). Moreover, we observed no changes in the expression of apoptosis-related genes in either the PANC-1 or the AsPC-1 cell lines [Kyoto Encyclopedia of Genes and Genomes

ASC knockdown reduces cyclin D1 (CCND1) level and stops the cell cycle at the G1 phase.
We then employed flow cytometry to determine whether ASC knockdown indeed affected the cell cycle in PDAC cells, with the results showing a substantial increase in the population of cells in the G1 phase and a concomitant reduction of cells in the S phase (Fig. 6a). Based on this result, we focused on CCNs that regulate the cell cycle in the G1 phase. Real-time RT-PCR revealed that expression of the G1-phase gene CCND1 was decreased by ASC silencing in PANC-1 cells and AsSPC-1 cells (Fig. 6b,c), whereas CCND2 expression was decreased in PANC-1 and undetected in AsPC-1 cells. Additionally, CCND3 expression increased in PANC-1 cells and decreased in AsPC-1 cells, and we observed no change in CCNE1 and CCNE2 expression in PANC-1 cells but slight reductions in AsPC-1 cells. Western blotting subsequently confirmed changes in CCND1 protein levels corresponding to the observed downregulation of gene expression in both cell lines (Fig. 6d). These results suggested that ASC-silenced cells were arrested in the G1 phase due to regulated expression of cell cycle-related genes, which subsequently decreased cell proliferation.

Downregulation of ASC level does not modulate the expression of apoptosis-related molecules in PDAC cells.
ASC is reportedly associated with apoptosis; therefore, we performed TUNEL staining and antibody arrays to observe changes in apoptosis in PDAC cells due to ASC downregulation. TUNEL staining revealed no changes in apoptosis upon ASC silencing at 3-and 5-days post-transfection with control and ASC siRNA (Figs. 7a,b; and S4). Furthermore, examination of apoptosis using antibody arrays revealed no changes in the expression of apoptosis-related proteins, including caspase-3, upon ASC downregulation (Fig. 7c,d).

Discussion
In recent years, increasing evidence has shown that ASC plays an important role in the interaction between PDAC cells and cells in the surrounding microenvironment (e.g., tumor-associated macrophages and cancer-associated fibroblasts) 27,28 ; however, the role of ASC in signal transduction in PDAC cells has not been fully investigated. The present study demonstrated that downregulation of ASC induced changes in cell cycle in PDAC cells and inhibited proliferation. A previous report identified methylated regions associated with ASC in 23% of papillary thyroid carcinomas and none in normal thyroid samples 29 . In other carcinomas, ASC methylation has been reported in 63.6% of prostate cancer, 40% of breast cancer cases, 32.1% of lung adenocarcinomas, 13.2% of lung squamous cell carcinomas, 38.5% of lung large-cell carcinomas, and 60% of lung small-cell carcinomas 10,29,30 . Although there are no studies on the frequency of ASC methylation in PDAC, a previous report indicates that > 90% of PDAC cells are IHC-positive for ASC 28 . In the present study, we identified ASC overexpression in human PDAC cells through bioinformatics methods. Additionally, IHC confirmed that ASC was more highly expressed in tumor tissues than in paired paracancerous tissues, as previously reported 28 . ASC expression is enhanced in ducts near the tumor and in metaplastic ducts and further enhanced in cancer, although rarely seen in ducts distant from  www.nature.com/scientificreports/ the tumor. This suggests that ASC may be involved in acinar-duct metaplasia, which contributes to the development of pancreatic cancer, and might be an important factor for carcinogenesis. Moreover, three of four PDAC cell lines exhibited ASC overexpression relative to levels observed in hTERT-HPNE cells generated from human pancreatic duct samples with acinar cells associated with the ductal metaplasia phenotype 31 . These results indicated that ASC expression might be suppressed by methylation in PDAC cell lines, such as MIA PACA-2; however, ASC is also considered to be highly expressed relative to levels observed in non-cancerous tissue. These results suggest that ASC methylation and expression vary according to the tissue and cancer type. Protti et al. 15 consider ASC as a tumor suppressor when its expression in tumor cells is lower than that in normal epithelial cells and non-tumor adjacent tissues and tumor-promoting when its expression in tumor cells is higher than that in normal tissue. In the present study, we found that elevated ASC mRNA expression in tumor tissues was a factor associated with poor prognosis for PDAC. Tumors with low mutational burden are reportedly less responsive to current immunotherapies and require new therapeutic targets. Therefore, ASC might represent a potential therapeutic target in tumors with low mutational burden, as we found that ASC expression is a prognostic factor for PDAC. In particular, it would by clinically important as a prognostic factor in grade 2 tumors, which are the most common in PDAC 32 . These findings suggest that ASC might be an essential biomarker of PDAC and contribute to PDAC progression.
There has been one report on ASC using PDAC cells. The results of this report showed that increased ASC expression due to demethylation in MIA PaCa-2 cells resulted in increased sensitivity to gemcitabine and docetaxel 33 ; however, there have been no studies reporting the detailed molecular function(s) of ASC in PDAC. Previous reports indicate that ASC plays an important role in apoptosis induction in various cancers 34,35 ; however, in the present study, we observed no induction of apoptosis following ASC silencing in PDAC cells. Additionally, in IMR90-E1A cells, ASC silencing significantly inhibited etoposide-induced apoptosis; however, in the absence of etoposide stimulation, there was no change in the percentage of apoptotic cells relative to controls 36 . These findings suggest that ASC does not affect apoptosis in pancreatic cancer cells, at least in the absence of exogenous apoptotic stimuli. On the other hand, ASC is also a key adaptor molecule of inflammasomes and activates procaspase-1, which is necessary for processing IL-1β and IL18 37,38 . In the present study, we found that www.nature.com/scientificreports/ IL-1β level in the supernatant was below the measurement sensitivity in PANC-1 and AsPC-1 cells (Fig. S5). Moreover, RNA-seq showed that ASC silencing did not induce changes in inflammasome-related gene groups (data not shown). Therefore, these findings suggest that ASC does not contribute to changes in apoptosis or inflammasome formation in PDAC cells. We observed suppression of PDAC cell proliferation following ASC silencing; however, this was not observed in MIA PACA-2 cells with poor ASC expression, suggesting that this is not an off-target effect. Additionally, because ASC-related effects were not observed in hTERT-HPNE cells, this suggests that ASC-specific effects are unique to PDAC cells. A previous report demonstrated that ASC silencing resulted in reduced cell viability, suppressed tumor growth, and arrested the cell cycle in the G1 phase in metastatic melanoma. The authors hypothesized that ASC might contribute to a positive feedback loop in cases of autoinflammation, where upregulated nuclear factor-κB expression results in transactivation of pro-IL-1β to promote autocrine IL-1 signaling in metastatic melanoma 13 . However, because no IL-1β was detected in the culture supernatant of PDAC cells in the present study and a previous report 39 , we speculated that other pathways are responsible for the changes in cell proliferation. Here, RNA-seq analysis showed that ASC silencing induced changes in the expression of cell cycle-related genes. The G0/G1 phase of the cell cycle is regulated by CCNs, including CCND and CCNE, among which we observed significant changes in only CCND1 expression. CCND1 and CCND3 are often differentially www.nature.com/scientificreports/ overexpressed in PDAC 40 , and CCND2 reportedly plays a role in the proliferation of pancreatic islet β-cells 41 , although its expression is infrequently detected in PDAC tissues and cells 40,42 . In the present study, ASC silencing www.nature.com/scientificreports/ induced a significant reduction in CCND1 expression in PANC-1 and AsPC-1 cells, whereas CCND3 expression increased in PANC-1 cells and decreased in AsPC-1 cells, and CCND2 was undetectable in AsPC-1 cells. Moreover, CCNE1 and CCNE2 expression did not show the same changes in PANC-1 and ASPC-1 cells. Based on these results, we speculated that CCND1 contributes to the change in cell proliferation observed following ASC silencing in PDAC cells. CCND1 expression is associated with anchorage-independent growth, tumorigenicity, angiogenesis, hypoxia response, and resistance to chemotherapeutic agents 43 . Furthermore, it plays an important role in G1/S-phase transition and cell proliferation 44 . Because ASC silencing resulted in an increase in the population of cells in the G1 phase and a concomitant reduction of cells in the S phase, we suggest that ASC regulates CCND1 expression and promotes G1/S-phase transition.
In summary, we found that downregulated ASC expression inhibited tumor progression by modulating the cell cycle in PDAC cells. These findings suggest ASC as a possible therapeutic target in PDAC; however, further studies are warranted to investigate this possibility.

Data availability
The datasets generated and/or analyzed during this study are available from the corresponding author upon reasonable request.